1. Field of the Invention
The invention is directed to methods for analyzing a microscope image comprising a plurality of picture elements, the intensity distributions of florescence events of a sample labeled with one or more types of fluorophores, and microscopes for implementing methods of this kind.
2. Description of Related Art
Research fields such as system biology which aim to describe biological systems by quantitative models rely on quantitative data. Such data could be determined heretofore only with poor accuracy by means of fluorescence microscopy, for example, by means of a “flying spot” microscope (Pawley, “Biological Confocal Microscopy”, Springer Verlag, 3rd edition 2006, page 6) or by calcium imaging (Pawley, page 529) because a large number of error sources and noise sources leads to fluctuations in intensity. Examples of error sources include the large variance in the Poisson distribution for fluorescence events, uncertainty about the size of the microscope detection volume, detector noise, photophysical properties such as quantum efficiency, excitation and efficiency spectra, and the bleaching behavior of the fluorophores. Other uncertainties include differences between different types of fluorophores, inhomogeneities in illumination, and background fluorescence and autofluorescence in the sample. These errors become larger (or more important) the smaller the volumes under examination and, therefore, the smaller the quantity of molecules examined and the higher the spatial resolving power of the microscope.
However, a high resolving power of the microscope being used is required for high spatial accuracy. The resolving power depends upon the so-called point spread function (PSF) of the microscope objective which always has a finite width (spatially, a finite volume) owing to the diffraction of the light received from the sample in the microscope objective, so that a point light source such as a fluorescing molecule is optically imaged on a finite surface. Therefore, the resolving power of a microscope is inherently limited (Abbe, 1873). However, a number of approaches are known from the prior art for generating images of a higher resolution than that allowed by these inherent limits (hereinafter referred to as high resolution).
For example, through a structured illumination of the sample in a variety of phase positions (structured illumination microscopy or SIM), the maximum resolving power can be improved laterally and axially approximately by a factor of two, or even more when combined with nonlinear excitation (saturated pattern excitation microscopy or SPEM). For this purpose, a result image of correspondingly higher resolution must be reconstructed computationally from the individual images recorded successively at the phase positions. SIM is disclosed, for example, in U.S. Pat. No. 5,671,085. SPEM is disclosed, for example, in U.S. Pat. No. 6,909,105.
The use of photoswitchable fluorescent dyes to increase resolving power (photoactivated localization microscopy; PALM or PAL-M) is known from WO 2006/127692 A2. An extremely small quantity of randomly distributed fluorescent dye molecules (fluorophores) is transformed (activated) to an excitable state by light of very low intensity at an activation wavelength and is subsequently excited to fluorescence in a known manner by light in an excitation wavelength. The rest of the fluorophores which are not activated cannot be excited to fluorescence by the excitation wavelength. Owing to the random distribution, the activated and excited fluorophores generally lie far enough apart from one another spatially so that there is no mutual overlapping of the intensity distributions of the point source images, which intensity distributions arise from the fluorescence events and are widened in a diffraction-limited manner. This is also true in particular for a projection on a two-dimensional image in which the intensity distributions inevitably extend over a plurality of picture elements (pixels) because of the diffraction widening. In PAL, microscopy, a multitude of individual images are acquired, each having a small quantity of generally non-overlapping fluorescence events. in so doing, the activation of a small group of fluorophores is not repeated until after the last fluorophores activated are photobleached. The origins of the individual fluorescence events are localized in the individual images based on the diffraction-broadened intensity distributions by means of a compensation computation with subpixel resolution and are entered in a high-resolution result image.
Other variants of individual fluorophore localization are known which differ in their approach to isolation. In STOR microscopy (stochastic optical reconstruction microscopy, or STORM), the fluorophores are switched back to their initial state (deactivated), for example, by means of a second light source, as soon as enough photons have been recorded in an individual image. Besides this, there are also the methods of PALMIRA (PALM with independently running acquisition), FPALM (fluorescence PALM), dSTORM (direct STORM), and GSDIM (ground state depletion and individual molecule return).
Most of the known high-resolution methods have the disadvantage that determining a highly resolved result image from the series of successively acquired normal-resolution individual images is extremely computation intensive and, therefore, time-consuming. Further, analysis of the result images which are generated is generally carried out visually by a human observer. In so doing, subjective influences can result in a broad dispersion when determining physical, biological or chemical quantities.